A. Field of the Invention
The present invention relates to a polypeptide termed ply_pitti26 comprising the sequence as depicted in SEQ ID NO:1 as well as variants of this polypeptide. Furthermore, the present invention relates to nucleic acids and vectors encoding for the polypeptide and variants thereof as well as host cells comprising these nucleic acids and/or vectors. Finally, the present invention relates to the uses of the polypeptide, variants thereof, nucleic acid sequences, vectors and host cells, in particular for the treatment or prophylaxis of a subject infected by or exposed to Staphylococci.
B. Background of the Invention
1. Bacterial Infections
Staphylococcal infections are a major cause of severe diseases with high mortality all over the world. The gram-positive pathogen Staphylococcus aureus is responsible for a variety of infections of the skin and soft tissues as well as life-threatening infections like bacteremia and endocarditis. In addition, Staphylococcus aureus is frequently involved in food poisoning. Due to its tolerance to low pH values and high salt conditions this pathogen grows in a variety of food products, especially of animal origin, producing a heat stable enterotoxin. Persons with a particular risk of infection are patients after surgery or during hemodialysis as well as premature infants and immunocompromised persons, or those with need for prosthetic devices. Staphylococcal infections are of particular global health concern because of their high distribution (about 25-30% of the population are asymptomatic carriers) and of the increasing emergence of antibiotic resistant strains of Staphylococcus aureus. MRSA (methicillin-resistant Staphylococcus aureus) is a prominent member of this group and a major cause of nosocomial infections. In addition, there are many multiresistant strains, even those which are resistant to the “drugs of the last line of defence” like vancomycin, linezolid or daptomycin. Infections with antibiotic resistant staphylococci rise enormous costs to the global health budgets, because the patients often need long-term stay in a hospital and have to be isolated from other patients.
Besides the coagulase-positive S. aureus, pathogens from the group of the coagulase-negative staphylococci are of importance. S. haemolyticus, for example, causes keratitis, S. epidermidis is frequently found in biofilms on implanted devices which are associated with serious infections (endoplastitis) and S. saprophyticus is responsible for urinary tract infections. Apart from infections of humans, cattle infections also play an important role. Especially, bovine mastitis, an infection of the mammary glands, is of commercial significance. Apart from S. aureus it is caused by some coagulase-negative staphylococci like S. epidermidis, S. simulans, S. chromogenes, S. hyicus, S. warneri and S. xylosus. 
Standard antibiotic therapy is becoming more and more ineffective. Hence, new strategies for treating bacterial infections are needed. They include the development of new antibiotics as well as the search for antimicrobial peptides. Uses of antibodies and putative vaccines or phage therapy are alternative approaches. However all of these methods exhibit serious disadvantages. At widespread use, novel antibiotics also will rise new resistances, antimicrobial peptides and monoclonal antibodies require a lot of additional investments until a routine use in therapy will be possible; immunization strategies against Staphylococcus aureus were not successful so far and phage therapy causes problems with immune response and tissue penetration as well as with a potential undesired transfer of bacterial toxins by the phages. The use of isolated peptidoglycan hydrolases, the so-called endolysins, represents an advancement of the phage therapy. Endolysins enzymatically hydrolyse the cell walls of those bacteria which are host organisms for their corresponding bacteriophages.
2. Bacterial Endolysins
After infection of the host bacterium, bacteriophages produce new phage particles within the host cell. At the end of the reproduction cycle the host cell must be lysed, to set free the new phage generation. Endolysins are produced as a tool for this lysis of the host cell. It was found, that endolysins also act on bacterial cell walls when they are added exogenously to non infected bacterial cells (“lysis from without”). The use of endolysins to kill contaminating bacteria in food was first disclosed by Gasson in 1991 (GB 2,255,561). First therapeutic and prophylactic applications in vivo using mouse model systems were described in 2001 by the group of Fischetti (Nelson & Fischetti, 2001; Loeffler et al., 2001). This work describes the topical application of endolysins against group A streptococci (oral application) and against pneumococci (nasopharyngeal application). Later, an application against Bacillus anthracis was added (Schuch et al., 2002). Entenza et al. (2005) report the use of Cpl-1 lysin against pneumococci causing endocarditis in rats. Endolysin PlyGBS was used to kill group B streptococci in the vagina and oropharynx of a mouse model (Cheng et al., 2005). Fischetti (2006) summarizes the use of phage lytic enzymes to control pathogenic bacteria.
In U.S. Pat. No. 5,997,862 a multitude of methods of treatments and pharmaceutical compositions to treat and prevent bacterial infections using phage derived lysins is disclosed. Several further patents teach specific compositions and uses of phage derived lysins for treatment of, e.g., dermatological infections, ocular infections, infections of mouth and teeth, infections of the respiratory tract, various illnesses, bacterial infections in general, the parenteral use of lysin compositions, and the use of bandage compositions. U.S. Patent Publication No. 2007/077235 describes lysin compositions to treat mastitis in animals.
Endolysins may be divided into five classes: (1) N-acetylmuramidases (lysozymes), (2) endo-β-N-acetylglucosaminidases, and (3) lytic transglycosylases, which all cleave the sugar moiety of peptidoglycan, (4) endopeptidases, which cleave the peptide moiety, and (5) N-actylmuramoyl-L-alanine amidases, which cut the amide bond between sugar backbone and peptide linkers. Endolysins show a modular organization exhibiting a combination of different polypeptide domains showing enzymatic activity or cell binding activity, the so-called EADs (enzymatically active domains) and CBDs (cell binding domains), respectively. Mostly, EADs are located at the N-terminal part of the endolysins, and CBDs at the C-terminal parts, but there are also exceptions of this rule of thumb. It is also shown that modules can be exchanged between different cell wall lytic enzymes producing new functional enzymes, which sometimes exhibit even new functional properties (Diaz et al., 1990; Croux et al., 1993; Donovan et al., 2006).
Since endolysins are typically more specific than antibiotics, it is unlikely that resistance development will rapidly occur. Therefore, the use of suitable endolysins acting on staphylococcus bacteria is a desirable means for the fight against the respective infections. Several endolysins active against staphylococcus bacteria are already described in the relevant art. Protein 17 associated with phage P68 is a staphylococcal endolysin which exhibits antimicrobial activity also against clinical S. aureus isolates (Takac et al., 2005). The endolysin plyTW derived from the S. aureus phage Twort needs only the N-terminal enzymatically active fragment for hydrolytic activity against bacterial cells, whereas the C-terminal part with homology to lysostaphin seems dispensable (Loessner et al., 1998). Donovan et al. (2006) created a chimaeric endolysin between Streptococcus agalactiae B30 endolysin and lysostaphin of Staphylococcus simulans with potential use in treatment of mastitis. Several groups used the endolysin of Staphylococcus aureus bacteriophage phi11 in antimicrobial applications. Navarre et al. (1999) identified multiple enzymatic activities in phi11 endolysin, and showed that a mutant with deletion of the amidase domain is still active. Donovan et al. (2006) used complete phi11 endolysin as well as C-terminally truncated versions in assays against mastitis pathogens. Different mutants of phi11 endolysin and phi12 endolysin were tested in different activity assays on Staphylococcus aureus cell walls, heat inactivated cells and also bacterial biofilms (Sass & Bierbaum, 2007). The endolysin of the Staphylococcus warneri phage ΦWMY, LysWMY, although reported to be closely related to phi11 endolysin, retained full activity when the amidase as well as the cell binding domains were deleted (Yokoi et al., 2005). This result indicates that the functions of and interactions between the different endolysin modules are not equivalent even in closely related endolysins.
Although different endolysins against staphylococcus bacteria are known from the art, there is still a need for efficient staphylococcal endolysins that can be produced in an efficient way and in addition show high activity against microorganisms of the genus Staphylococcus. 